Isolation and Characterization of Zearalenone Sulfate Produced by Fusarium spp.t

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jn. 1991, p Vol. 57, No /91/1146-5$2./ Copyright C) 1991, Amerin Soiety for Mirobiology Isoltion nd Chrteriztion of Zerlenone Sulfte Produed by Fusrium spp.t JAVIER PLASENCIA AND CHESTER J. MIROCHA* Deprtment of Plnt Pthology, University of Minnesot, St. Pul, Minnesot 5518 Reeived 2 August 199/Aepted 23 Otober 199 A wter-soluble ompound relted to zerlenone ws isolted from ulture of Fusrium grminerum 3 grown in rie. The struture of the novel metbolite ws determined to be zerlenone-4-sulfte on the bsis of fst-tom-bombrdment mss spetrometry, proton nuler mgneti resonne, UV spetrosopy, nd by hemil nd enzymti retions. Strins representing Fusrium equiseti, Fusrium smbuinum, nd Fusrium roseum produed the sulfte onjugte s well. In the rt uterus enlrgement biossy, the metbolite or its hydrolysis produt ws found to retin the estrogeni tivity hrteristi of zerlenone. Nturl ourrene of this novel metbolite might be signifint beuse nlytil methods devised for zerlenone in grin nnot detet the onjugte but the onjugte retins the biologil properties of the myotoxin when ingested by nimls. Zerlenone, myotoxin produed by severl Fusrium spp., is frequently found to ontminte vrious erel grins (3) nd other ommodities. A reent worldwide survey (24) reports zerlenone ourring in 58% (n = 45) of the orn smples olleted in 19 different ountries. The estrogeni syndrome triggered by zerlenone when it is fed to swine (7) inludes diminished or nonfertility, prolonged estrus, redued litter size, mlformtions in offspring, nd juvenile hyperestrogenism. Zerlenone hs lso been implited in deresed fertility ses in ows fed moldy hy (15). Some strins of Fusrium spp. produe, in ddition to zerlenone, other relted metbolites suh s lphzerlenol, whih hs lso been found in feeds (18). The distereomers of 8'-hydroxyzerlenone (13) nd of 3'-hydroxyzerlenone (2) hve been isolted nd hrterized from ultures of Fusrium roseum. Another soure for the study of zerlenone derivtives is the fluids of nimls tht hve been dosed with the myotoxin. Zerlenone is found s its gluuronide ddut in the urine of ows, rts, rbbits, nd swine. The sulfte onjugte is lso reported to our in ow urine s determined indiretly by enzymti hydrolysis nd then nlysis of zerlenone (17). However, the identity of the onjugte ws not onfirmed. In the present study we report the isoltion of the zerlenone sulfte onjugte from rie ulture of Fusrium grminerum. The hemil, spetrl, hromtogrphi, nd biologil properties of this novel derivtive of zerlenone re desribed. MATERIALS AND METHODS Isoltion of the metbolite. F. grminerum 3 ws originlly isolted from orn stlk in Minnesot (26) nd when grown in utolved rie produed lrge mounts (1,5 mg/kg) of zerlenone (27). The ground moldy rie (1 g) ws pled into 1-liter Erlenmeyer flsk nd moistened with enough wter to form pste. A 2-ml volume of * Corresponding uthor. t Pper no. 18,424 from the Minnesot Agriulturl Experiment Sttion. methnol-wter (3:1, vol/vol) ws dded, nd the flsk ws shken for 1 h in wrist-tion shker. The extrt ws filtered through Whtmn no. 4 filter pper into 1-liter seprtory funnel. The residue ws reextrted twie with 2 ml of methnol-wter, nd the filtrtes were pooled in the funnel. Sturted sodium hloride solution (2 ml) ws dded to the funnel, nd the extrt ws deftted by prtitioning it twie with 3 ml of petroleum ether. The queous methnoli extrt ws poured into round-bottomed flsk, nd bout 8% of the methnol ws evported in rotry evportor t 45 to 5 C. The remining solution ws diluted volume to volume with distilled wter nd prtitioned with ethyl ette (three times), whih ws olleted nd evported to dryness in rotry evportor. The residue ws redissolved in 2 ml of hloroform-methnol (9:1, vol/vol) nd loded into glss olumn (2 m by 1.5 m [inside dimeter]) pked with 2 g of sili gel (1 to 2 mesh). The sili gel olumn ws previously equilibrted with hloroform-methnol (98:2, vol/vol). The olumn ws eluted suessively with 5 ml of eh one of the following mixtures of hloroform-methnol: 9:1, 8:2, nd 6:4 (vol/vol). Frtions of 5 to 7 ml were olleted nd sreened on sili gel-thinlyer-hromtogrphy (TLC) pltes whih were developed with hloroform-methnol (8:2, vol/vol), nd the zerlenone derivtive ws mde visible under short-wvelength UV light. Frtions ontining n intense fluoresent spot with n Rf vlue of.25 were pooled in round-bottomed flsk nd onentrted to dryness in rotry evportor. The residue ws redissolved in.35 ml of methnol nd pplied on sili gel-tlc pltes (2 by 2 m,.25-mm thikness; Merk) whih were developed in hloroformmethnol (8:2, vol/vol). The fluoresent bnd with n Rf of.25 ws srped off the pltes nd eluted with hloroformmethnol (6:4, vol/vol). The solvent ws evported, nd the residue ws rystllized with benzene nd then dried with vuum t room temperture overnight. About 2 mg of yellow powder ws obtined nd then used for further hemil nd biologil tests. FAB mss spetrometry. Negtive-ioniztion fst-tombombrdment (FAB) mss spetrometry ws rried out in VG77EQ mss spetrometer. The ompound ws dis- 146 Downloded from on My 2, 218 by guest

2 57, 1991 solved in methnol, nd 5,lI of the solution ws mixed with the thioglyerol mtrix on the probe. A resolution of 2,5 ws used in obtining n urte mss mesurement. Referene msses were m/z 413 nd 369 from polyethylene glyol. Chemil nd id hydrolysis. One milligrm of the isolted ompound ws treted seprtely with 1% hydrohlori id nd sulftse from limpets (Sigm Chemil Co., St. Louis, Mo.). Aid hydrolysis ws rried out t 65 C for 4 h, while enzymti hydrolysis ws performed in wter bth t 37 C for 4 h. When the retion ws ompleted, the retion mixtures were extrted with methylene hloride nd both the orgni nd queous frtions were nlyzed; the queous-phse residue ws redissolved in.1 ml of wter nd treted with 3 drops of 3% solution of brium hloride. The orgni-lyer residue ws nlyzed for zerlenone by TLC nd ompred with uthenti zerlenone stndrd. For further onfirmtion, the orgni lyer ws evported to dryness nd reted with the derivtiztion regent BT (Piere Chemil Co., Rokford, Ill.) for the formtion of the trimethylsilyl ether. The derivtive ws nlyzed by gs hromtogrphy-mss spetrometry nd ompred with uthenti zerlenone-trimethylsilyl. UV spetrosopy. Zerlenone nd the isolted ompound were dissolved in methnol, nd their UV bsorption spetr were reorded on Bekmn DB-GT grting spetrophotometer. NMR spetrosopy. The proton-nuler mgneti resonne (NMR) spetrum of the ompound ws reorded in 3-MHz Niolet NT-3-W3 spetrometer by using tetrmethylsilne s the internl stndrd nd CD3D s solvent. Biologil tivity. The estrogeni tivity of the ompound ws ompred to tht of zerlenone in the rt uterus enlrgement biossy. Eighteen Sprgue-Dwley 2-dy-old virgin rts (Bio-Lb Corp., St. Pul, Minn.) were divided rndomly into three groups of six rts eh nd housed in the Reserh Animl Resoures fility, University of Minnesot, St. Pul, Minn. Equivlent molr mounts (63 nmol) of zerlenone or zerlenone sulfte (s lulted by the moleulr weight of the nion, i.e., 397) were dministered intrgstrilly to eh rt in.5 ml of the rrier solvent (2% ethnol in sterile wter). A ontrol group ws dosed with the rrier solvent. Twenty-four hours fter gstri intubtion, the nimls were weighed nd killed by ervil dislotion. The nimls were disseted, nd the uteri were removed nd weighed in n nlytil blne. The rtio of the uterus weight to totl body weight ws reorded for eh niml. The men nd stndrd devition were lulted for eh group, nd dt were nlyzed by using the Student's t test. Prodution of zerlenone sulfte by Fusrium spp. Zerlenone sulfte prodution ws determined by TLC nlysis of the extrt of rie in whih isoltes representing vrious speies known to produe zerlenone were grown. For this nlysis, 2 g of rie ws extrted with 12 ml of methnol-wter (3:1, vol/vol) nd deftted s desribed in the isoltion proedure. The wter-methnol frtion ws onentrted to evporte most of the methnol, nd the queous phse ws then prtitioned with ethyl ette (5 ml, three times). The orgni lyer ws dried, redissolved in 1 ml of methnol, nd spotted on the TLC plte whih ws ospotted with purified zerlenone sulfte. Quntittive nlysis of the ompound ws done by high-pressure liquid hromtogrphy by using Cl8 reverse-phse olumn with wter-etonitrile-methnol (2:1.6:1, vol/vol/vol) s mobile ZEARALENONE SULFATE PRODUCTION BY FUSARIUM SPP. OW U) - 5 i IN1 11 o m 256 L, LhiiA.ii lli. 317 U UEPHII I 1ee s FIG. 1. Negtive-ioniztion FAB mss spetrum of the isolted metbolite in thioglyerol mtrix. Moleulr nion (M-H)-, m/z 397, loses the sulfoni group to yield m/z 317 whih orresponds to zerlenone moleulr weight minus proton. Pek t mlz 419 orresponds to the sodium ddut of the moleulr ion. phse nd fluoresene detetion (exittion wvelength, 274 nm; emission wvelength, 418 nm). RESULTS 1- Tretment of the ompound with 1% hydrohlori id yielded zerlenone whih ws identified in the methylene hloride lyer by norml-phse TLC (Rf.77 in hloroform-methnol [9:1, vol/vol]) nd by the eletron-impt = mss spetrum of the trimethylsilyl ether derivtive. The dignosti ions of zerlenone-trimethylsilyl ether were found s desribed by Miroh et l. (16): M+ 462, 447, 429, 333, 35, 26, nd 151. The wter-soluble frtion produed white preipitte of brium sulfte when it ws reted with 3% queous solution of brium hloride. Sulftse tretment of the ompound lso gve zerlenone s the glyone nd sulfte ion whih preipitted when treted with brium hloride. The negtive-ioniztion FAB mss spetrum of zerlenone sulfte (Fig. 1) showed three mjor peks: the moleulr nion (M-H)- t m/z 397 loses the sulfoni group (SO3f) to yield mlz of 317 (M-SO3-H)-. The pek with m/z 419 orresponds to (M-H + N)-. The empiril formul proposed for the ompound ws (Cl8H218S) orresponding to lulted mss of nd ws onfirmed by ext mss mesurement with negtive-ioniztion FAB mss spetrometry. The spetrum reveled the ion with n observed mss of whih differs from the lulted empiril formul by only 2.2 millimss units. The UV spetrum (Fig. 2) of the zerlenone onjugte in methnol reveled n ppreible shift in two bnds. The mximum bsorption bnd of 236 nm in zerlenone is shifted to 228 nm in the onjugte, wheres the bnd t 274 in the prent ompound is observed t 26 nm in the onjugte. These observed shifts re ttributed to the eletron-withdrwing effet of the sulfte group (2). The 'H-NMR spetrum (Tble 1) shows tht the phenoli hydroxyl group t C-4 observed t 9.3 ppm in zerlenone is not present in zerlenone sulfte, supporting the ssignment of the position of the sulfte ester t C-4 in zerlenone. The novel ompound isolted from the F. grminerum ulture ws identified s zerlenone-4-sulfte (Fig. 3) on the bsis of mss spetrometry, hemil nd enzymti re- 147 Downloded from on My 2, 218 by guest

3 148 PLASENCIA AND MIROCHA APPL. ENVIRON. MICROBIOL. ) 4 ID.8 F.61-.4O Wuelength (nm) FIG. 2. UV bsorption spetrum of zerlenone ( ) nd of the isolted metbolite (-- --) in methnol. tions, nd UV nd NMR spetrosopies. The metbolite is soluble in wter, methnol, ethnol, nd ethyl ette, slightly soluble in hloroform, nd insoluble in hexne, benzene, nd petroleum ether. In norml-phse TLC, the metbolite hs n Rf vlue of.25 (hloroform-methnol, 8:2, vol/vol). In reverse-phse high-pressure liquid hromtogrphy, by using the mobile phse indited for zerlenone nlysis, it elutes t 1.42 min (Fig. 4) from 25-m-long C18 olumn. Although the retention time is lose to tht of the solvent front, dequte quntifition of its onentrtion in ultures ws possible by using the purified metbolite TABLE 1. I14 236' 228,1 II II l I, 1. 'H-NMR hemil shifts of zerlenone nd its metbolite in CD3OD Chemil shift of: Position Zerlenone Zerlenone sulfte ppm (J=Hz) ppm (J=Hz) 2-OH H (1.4) 6.9 (1.4) 4-OH 9.3 H (1.3) 6.8 (1.3) H-i' 7.1 (1.4) 6.92 (1.4) H-2' 6.21 (1.2) 6.77 (1.3) H-3' H-4' H-5' H-6' H-7' H-8' H-9' H-1' 5.3 (1.) 5.4 (1.) 11'-CH I I 11'' $ 1': 26 ' I I I 27 ' I FIG. 3. Chemil struture of zerlenone-4-sulfte. s stndrd. Beuse onjugtion dds 79 mss units to the moleulr weight of zerlenone, the molr onentrtion ws used for omprison. The prodution of zerlenone nd zerlenone sulfte by F. grminerum 3 over 3-dy period follows similr pttern, mintining onstnt molr rtio (Fig. 5). Four different Fusrium speies were found to produe the metbolite (Tble 2) when grown in rie substrte. Conentrtions rnging from 3 nmol/g of rie (123 ppm) to 58 nmol/g of rie (23 ppm) were found in the five ultures tested. Molr rtios of zerlenone to zerlenone sulfte vried from 12:1 to 2:1. The rt uterus enlrgement biossy (Fig. 6) showed tht when zerlenone sulfte is ingested by rts, it prtilly retins the estrogeni properties of the prent ompound. DISCUSSION The identity of the wter-soluble metbolite isolted from F. grminerum 3 ws estblished s zerlenone-4-sulfte by using the riteri set by nturl produts hemists in the systemti identifition of flvonoid sulftes (2). Sulfted metbolites hve been widely studied in mmmls nd other niml speies s end produts of the metbolism of xenobiotis (5, 22). In the plnt kingdom, sulfted produts, minly flvonoids, hve been isolted from more thn 25 speies, inluding diotyledons nd monootyledons (2). In fungi, however, few studies of sulfte onjugtes pper in the literture. Choline sulfte is produed by Aspergillus nidulns presumbly s sulfur reserve (12). Studies by Cernigli et l. (6) report the sulfte onjugtion of romti hydrorbons in liquid fermenttion by Cunninghmell elegns. Although only limited survey mong our isoltes ws done, we were ble to detet zerlenone sulfte in four speies of Fusrium, ll of them known to produe the prent myotoxin. The rtio of zerlenone to zerlenone sulfte is high ompred with the prodution of other zerlenone derivtives (21). In the mmmlin systems, the ondenstion with sulfte ours vi the formtion of n tivted sulfur donor, 3'-phosphodenosine-5'-phosphosulfte, whih is onjugted with n lohol group by sulfotrnsferse (5). Although the retion is primrily used for phenols, other hydroxyl groups n be sulfted s well. The bility to onjugte phenols nd lkyl lohols in Fusrium spp. hs been demonstrted in this nd one other study, lthough the mehnism of onjugtion is unknown. Sulfte onjugtion in Fusrium spp. hs been reported by Vesonder et l. (25), who found sterol sulfte in orn ultures of F. grminerum. In ddition to zerlenone nd sterols, Fusrium spp. produe other myotoxins, suh s trihotheenes nd Downloded from on My 2, 218 by guest

4 VOL. 57, 1991 ZEARALENONE SULFATE PRODUCTION BY FUSARIUM SPP. 149 (u Zerlenone sulfte Cw zerlenone Time (min) FIG. 4. Elution profile of zerlenone nd zerlenone sulfte seprted on p.bondpk C18 olumn by using methnol-etonitrile-wter (1:1.6:2, vol/vol/vol) s mobile phse nd fluoresene monitor for detetion. Retention time of eh metbolite is indited t the top of the pek. fumonisins, whih lso ontin hydroxyl groups tht n be onjugted with sulfte esters. Other orgnisms, when inubted in vitro with zerlenone, re ble to onjugte the myotoxin with D-gluose; Rhizopus sp. (14), Muor binieri (9), nd orn ells in tissue ulture (1) form the 4--bet-gluoside onjugte, wheres Thmnidium elegns produes the 2,4--bet-digluoside (8). Greis et l. (11) report the nturl ourrene of zerlenone gluoside in brley nd whet, suggesting tht the myotoxin is being onjugted by the plnt ells. Although this might our, onjugtion of zerlenone by Fusrium spp. is possible. This is the first report on the prodution of zerlenone onjugte by the sme orgnism responsible for zerlenone synthesis. A noteworthy differene between gluose nd sulfte onjugtes is their reltive stbility to id hydrolysis. While gluosides re very resistnt to id, whih might explin their lk of tivity in swine (11), ryl sulfte esters re redily hydrolyzed by id (23). Zerlenone sulfte is lso suseptible to id hydrolysis; therefore, zerlenone is relesed when the onjugte is ingested by nimls. Intestinl bsorption into the bloodstrem might explin how, in the rt uterus enlrgement biossy (Fig. 6), the onjugted zerlenone triggered n estrogeni response hrteristi of the myotoxin. The presene of zerlenone sulfte in feeds might explin m E -p r- e zerlenone I^* - -- * zerlenone sulfte Time (dys) FIG. 5. Prodution of zerlenone nd zerlenone sulfte by F. grminerum 3 grown in rie t 25 C over 3-dy period. Both metbolites were quntified by high-pressure liquid hromtogrphy. the estrogeni syndrome in the field in whih zerlenone is identified t low onentrtions, but it is not enough to explin ll of the symptoms observed in the nimls. The onjugtion of n orgni moleule with the sulfte ion results in importnt hnges in the hemil nd physil properties of the prent ompound. Minly, the solubility in wter nd idity inreses. As detoxifition mehnism in mmmls, inresed wter solubility implies filitted urinry nd biliry exretion (5). From n nlytil point of view, inresed wter solubility implies tht the sulfte onjugte of zerlenone nnot be extrted nd nlyzed by nlytil methods urrently devised for zerlenone. Chloroform extrtion, id-bse prtition lenup, nd seprtion by reverse-phse high-pressure liquid hromtogrphy s desribed in the urrent offiil method of the Assoition of Offiil Anlytil Chemists for nlysis of zerlenone nd lph-zerlenol (4) nnot detet the sulfte onjugte. Figure 4 shows tht, by the retention time t whih the onjugte elutes, the onjugte would be msked by omponents of the orn mtrix in routine nlysis. Zerlenone sulfte hs not yet been deteted in feeds. Current methodology for nlyzing zerlenone onjugtes in serum nd urine (17, 19) uses indiret tehniques for the detetion of sulfte dduts; the smple is treted with hydrolyti enzymes nd the free zerlenone is quntified. For demonstrtion of the nturl ourrene of zerlenone onjugtes, similr pproh n be followed in feed nlyses. However, relible tehniques for the diret determintion of zerlenone onjugtes should be developed to ssess the totl mount of zerlenone to whih humns nd nimls re exposed. TABLE 2. Prodution of zerlenone nd zerlenone sulfte in rie ulture by vrious Fusrium spp. Amt produed Fusrium speies Refer- (nmol/g of rie) Molr nd ode ene Zerlenone rtio Zerlenone sulfte F. grminerum ,4 57 4:1 F. grminerum 1 1 3, :1 F. equiseti 2 1 3,7 3 12:1 F. smbuinum N45B l :1 F. roseum "gibbosum" 2 5, 58 1:1 Downloded from on My 2, 218 by guest

5 15 PLASENCIA AND MIROCHA E b n = 6 zerlenone zoo-sulfte 2%-ethnol FIG. 6. Uterotropi tivity of equimolr doses of zerlenone nd zerlenone sulfte in the rt uterus enlrgement biossy. The third group ws dosed with the rrier solvent (2% ethnol). Brs represent men of six rts per group plus stndrd devition. Different letters t the top indite differenes t P <.5. ACKNOWLEDGMENTS We thnk Wen Jo-Cheong for the ext FAB mss spetrometry nlysis nd interprettion. This work ws supported by Crgill Reserh Foundtion grnt (Projet 22-34H). REFERENCES 1. Abbs, H. K., U. Bosh, nd C. J. Miroh Abstr. Annu. Meet. Am. Phytopthol. So Phytopthology 78:1552. l.abbs, H. K., C. J. Miroh, B. P. Berdl, L. Sundheim, R. Gunther, nd B. Johnsen Isoltion nd toxiity of Fusrjum speies from vrious res of Norwy. At Agri. Snd. 37: Brron, D., L. Vrin, R. K. Ibrhim, J. B. Hrborne, nd C. A. Willims Sulphted flvonoids-n updte. Phytohemistry 27: Bennett, G. A., nd. L. Shotwell Zerlenone in erel grins. J. Am. Oil Chem. So. 56: Bennett, G. A.,. L. Shotwell, nd W. F. Kwolek Liquid hromtogrphi determintion of lph-zerlenol nd zerlenone in orn: ollbortive study. J. Asso. Off. Anl. Chem. 68: Cldwell, J Conjugtion retions, p In P. Jenner nd B. Test (ed.), Conepts in drug metbolism, prt A. Mrel Dekker, In., New York. 6. Cernigli, C. E., J. P. Freemn, nd R. K. Mithum Gluuronide nd sulfte onjugtion in the fungl metbolism of romti hydrorbons. Appl. Environ. Mirobiol. 43: Chng, K., J. H. Hurtz, nd C. J. Miroh Effets of the myotoxin zerlenone on swine reprodution. Am. J. Vet. Res. 4: El-Shrkwy, S. H Mirobil trnsformtion of zerlenone. III. Formtion of 2,4--bet-digluoside. At Phrm. Jugosl. 39: El-Shrkwy, S. H., nd Y. Abul-Hjj Mirobil trnsformtion of zerlenone. I. Formtion of zerlenone-4-o-betgluoside. J. Nt. Prod. 5: Engelhrdt, G., G. Zill, B. Wohner, nd P. R. Wllnofer Trnsformtion of the Fusrium myotoxin zerlenone in APPL. ENVIRON. MICROBIOL. mize ell suspension ultures. Nturwissenshften 75: Greis, M., J. Buer, J. Thiem, G. Plnk, S. Grbley, nd B. Gedek Clevge of zerlenone-glyoside, "msked" myotoxin during digestion in swine. J. Vet. Med. Ser. B 37: Hussey, C., B. A. Orsi, J. Sott, nd B. Spener Mehnism of holine sulphte utiliztion in fungi. Nture (London) 27: Jkson, R. A., S. W. Fenton, C. J. Miroh, nd G. Dvis Chrteriztion of two isomers of 8'-hydroxyzerlenone nd other derivtives of zerlenone. J. Agri. Food Chem. 22: Kmimur, H Conversion of zerlenone to zerlenone gluoside by Rhizopus sp. Appl. Environ. Mirobiol. 52: Miroh, C. J., J. Hrrison, nd A. A. Nihols Detetion of fungl estrogen (F-2) in hy ssoited with infertility in diry ttle. Appl. Mirobiol. 16: Miroh, C. J., S. V. Pthre, nd C. M. Christensen Zerlenone, p In J. V. Roderiks, C. W. Hesseltine, nd M. A. Mehlmn (ed.), Myotoxins in humn nd niml helth. Pthotox Publishers, Prk Forest South, Ill. 17. Miroh, C. J., S. V. Pthre, nd T. S. Robison Comprtive metbolism of zerlenone nd trnsmission into bovine milk. Food Cosmet. Toxiol. 19: Miroh, C. J., B. Shuerhmer, C. M. Christensen, M. L. Niku-Pvol, nd M. Nummi Inidene of zerlenol (Fusrium myotoxin) in niml feed. Appl. Environ. Mirobiol. 38: Olsen, M. E., H. I. Pettersson, K. A. Sndholm, nd K. H. Kiessling Quntittive liquid hromtogrphi method using fluoresene detetion for determining zerlenone nd its metbolites in blood plsm nd urine. J. Asso. Off. Anl. Chem. 68: Pthre, S. V., S. W. Fenton, nd C. J. Miroh '- Hydroxyzerlenones, two new metbolites produed by Fusrium roseum. Agri. Food Chem. 28: Rihrdson, K. E., W. M. Hgler, Jr., nd C. J. Miroh Prodution of zerlenone, lph- nd bet zerlenol by Fusrium spp. in rie ulture. J. Agri. Food Chem. 33: Roy, A. B Sulphte esters nd the sulphtses, p In G. J. Mulder, J. Cldwell, G. M. J. Vn Kempen, nd R. J. Vonk (ed.), Sulphte metbolism nd sulphte onjugtion. Tylor nd Frnis, London. 23. Roy, A. B., nd P. A. Trudinger The biohemistry of inorgni ompounds of sulphur, p Cmbridge University Press, Cmbridge. 24. Tnk, T., A. Hsegw, S. Ymmoto, U. S. Lee, Y. Sugigur, nd Y. Ueno Worldwide ontmintion of erels by the Fusrium myotoxins nivlenol, deoxynivlenol nd zerlenone. 1. Survey of 19 ountries. J. Agri. Food Chem. 36: Vesonder, R. F., H. R. Burmeister, nd L. W. Tjrks , 4,24 - trimethylholest- 8, 14, 24(28) - trien - 2lph, 3bet, lllph, 12bet-tetrol 12-ette, 3-sulphte from Fusrium grminerum. Phytohemistry 29: Windels, C. E., nd T. Kommedhl Lte oloniztion nd survivl of Fusrium grminerum group II in orn stlks in Minnesot. Plnt Dis. 68: Windels, C. E., C. J. Miroh, H. K. Abbs, nd W. Xie Peritheium prodution in Fusrium grminerum popultions nd lk of orreltion with zerlenone prodution. Myopthologi 81: Downloded from on My 2, 218 by guest